The field of the invention is spectrophotometry. Devices and methods of the invention are applicable to all uses of spectrophotometry, i.e., the measurement of light absorption or scattering in liquids, gases and solids, in addition to absorption, reflection, and scattering of light at interfaces. A wide range of spectroscopic and analytical instruments and devices may benefit from the invention. Exemplary applications of the invention include Ultra Violet-Visible (UV-Vis), Infrared (IR), Atomic Absorption (AA), circular dichroism (CD) spectrophotometers, and High Performance Liquid Chromatography (HPLC).
A fundamental property of a sample, be it gas, liquid or solid, is its tendency or lack of tendency to absorb or scatter light at certain wavelengths. Characterization of the tendency of a sample to absorb, scatter or transmit is the basis for spectrophotometry. Example applications include chemical and biological sample analysis. Other example applications include manufactured product testing and the testing of air or water quality.
The point of any application of quantitative spectrophotometry is the ability to numerically characterize a sample in order to discover sample properties or to differentiate it from another sample. Irrespective of the application, the critical aspects of quantitative spectrophotometry are sensitivity, precision, and accuracy. The sensitivity of a spectrophotometric measurement directly relates to the ability to detect small differences between samples having similar absorption properties. The greater the sensitivity, the smaller the difference that can be detected. The precision of a spectrophotometric measurement may be considered as a function of the ability to repeat the same measurement for an identical sample at different times. The accuracy of a spectrophotometric measurement may be considered as a function of the ability to correctly determine the numerical measure of the sample composition. The latter is critical, for example, when attempting to quantify an unknown element in a sample. Over a given range of concentration, the quantification is characterized by certain levels of precision and accuracy. However, below the lower limit of the concentration range, both precision and accuracy are adversely affected. This lower limit is the detection limit of the particular spectrophotometric instrument. As sensitivity increases, the detection limit decreases. Improvements in sensitivity, while retaining high levels of precision and accuracy are desirable.
One known application of spectrophotometry is spectrophotometric chemical analysis. Consideration of this technology is useful to illustrate the problems encountered when practical devices are used to measure light absorption. Spectrophotometric chemical analysis is a standard method for the determination of concentrations of light absorbing substances in liquids and gases. If solutions are studied, the substances are referred to as solutes. In practice, the quantity measured is the Absorbance (A), which is defined by the Beer-Lambert law as A=xe2x88x92log T, where T is the Transmittance. The Absorbance, which is given in Absorbance Units (AU), is proportional to C, the concentration of the absorbing substance by the relationship A=xcex5LC, where L is the length of the light path through the sample and xcex5 is a proportionality constant called the Absorptivity, which is specific to the absorbing substance. In order for the equations to be valid, terms A and T must relate only to absorption of light by the solute. Correction must be made for any interference, i.e., absorption other than that attributable to the solute. In practical devices, that type of interference can arise from various sources such as absorption/scattering attributable to the solvent or light reflected by portions of the device being used to measure absorption.
Spectrophotometers generally include a controlled optical system, a sample, detection system, and means for data analysis. The optical system produces a controlled beam or beams to pass through the sample or samples and then be collected by detectors. Detector outputs, which are proportional to the light powers, are then used for data analysis. A typical spectrophotometer has a dual beam optical system and is equipped with two cells, designated Sample and Reference. The power of light emerging from the cells results in detector currents, iS and iR, which are converted to voltages, VS and VR, respectively. For the precise measurement of A, interference corrections are performed by making two separate determinations. First, the ratio Q0=VS0/VR0 is determined with pure solvent in both S and R cells. Second, the ratio Q=VS/VR is determined with solution in the S cell and pure solvent in the R cell. Thus, one calculates T=Q/Q0 and A=xe2x88x92log T. Care must be taken when discussing the Absorbance because some systems give a response that is not identical to A as defined herein. Such a response may be useful as a qualitative indicator for monitoring purposes and it is often referred to as an xe2x80x9cAbsorbancexe2x80x9d. Absorbance values referred to in this application concern the absorbance values as defined by the Beer-Lambert Law, a quantitative measurement.
Others have recognized some sensitivity limits in spectrophotometry and some attempts have been made to reduce noise. Different spectrophotometric devices will have different limits. The sensitivity limits vary depending on the spectral region in question. Consider a UV-visible scanning instrument, of the type that is widely used for chemical analyses. This instrument uses a Tungsten lamp source to cover the visible range. The detectors are either photodiodes or photomultipliers. The generally accepted standard noise specification (Absorbance standard deviation) for high quality commercial units is "sgr"A=5xc3x9710xe2x88x925 AU (at 500 nm wavelength, 1 sec time constant). There is some misconception that this noise originates in the detectors as shot noise. However, with the use of a light meter equipped with a Silicon photodiode detector, it is easy to monitor the power output of a Tungsten lamp with a regulated power supply in a laboratory setting. Analysis of such results obtained by us shows that the Relative Noise Standard Deviation, "sgr"V/V, is about 5xc3x9710xe2x88x925, which (from the Beer Lambert Law) equals a noise level Standard Deviation of about 2xc3x9710xe2x88x925 AU, similar to the commercial noise level specification. Also, this noise is independent of the light power received by the detector in contrast to the basic characteristics of shot noise. Of course, other light source types will have different noise characteristics.
Furthermore, this noise level is about 100-fold greater than the calculated shot noise with detector current of 1-2 xcexcA, as in the present embodiments. Thus, source noise is a more important factor than detector shot noise in determining spectrophotometer sensitivity. That source noise limits performance was recognized by Haller and Hobbs. See, K. L. Haller and C. D. Hobbs, SPIE Vol. 1435, pp. 298-309 (1991).
Where source noise is determined to be dominant, steps can be taken to reduce the noise. Use is made of the fact that source noise is coherent in the two beams of a dual beam spectrophotometer, in which case, it is known that at least some of the noise can be canceled. Various noise cancellation circuits have been proposed. The detector circuit of Hobbs (U.S. Pat. No. 5,134,276) has been cited in the patent literature and elsewhere. Noise cancellation occurs because the source and reference currents are balanced at a node in the circuit. To accomplish this, the reference current is divided by use of a differential transistor pair that acts as a current splitter. The differential voltage controls fractions of current through the two legs of the current splitter across the transistor bases. Current balance can be achieved manually by applying an external differential voltage or it can be achieved automatically by use of a feedback loop to supply the differential voltage. The circuit has been used as a means to cancel laser noise both in communications and spectrophotometric applications.
The Hobbs circuit is also used for noise suppression in a capillary separation system, see Yeung et al., U.S. Pat. No. 5,540,825. A laser is used to monitor liquid flowing through a capillary, so that when a light absorbing substance enters the region of the capillary being monitored, it can be detected. A commercial detection system was replaced with the circuit of Hobbs, and noise reduction was obtained. A commercialized version of the Hobbs circuit is sold under the Trade Name xe2x80x9cNirvana.xe2x80x9d
To use the circuit of Hobbs, one measures the voltage output, LOGO=K ln(iR/iSxe2x88x921), where K is a proportionality constant. Thus knowing K, one can calculate Q=iS/iR from LOGO. K may be readily adjusted since it is determined by resistors in a voltage divider network. The automatic balance feature of the circuit makes it very convenient to use. However, there is one disadvantage that may not be immediately obvious, which has to do with the properties of the term ln(iR/iSxe2x88x921). This function becomes infinite as iRxe2x86x92iS, so that a sufficient imbalance in detector currents is necessary for proper operation. Depending upon the size of the imbalance, it may be necessary to measure small changes superimposed on relatively large voltages. For example, with electronic components values as given by Hobbs, and with a Tungsten source, and Silicon photodiode detectors, typical values for Reference and Sample detector currents are 2.5 xcexcA and 2 xcexcA, respectively, and LOGO is 1.470089 V. If the Sample current is reduced by 1 ppm (1 part in 106), LOGO is 1.470077 V. In this example, the numbers show LOGO must be determined to 7 significant figures (accuracy of a few parts in 107) in order to measure an Absorbance of 10xe2x88x926. This is a potentially significant disadvantage because of potential tracking errors and digitization requirements.
The term ln(iR/iSxe2x88x921) becomes zero as iRxe2x86x922 iS, so that it is possible to make LOGO small by adjusting the beam intensities to a ratio of 2:1. This is readily accomplished when measurements are made at a single wavelength or over a very small wavelength range, as with laser studies of Haller and Hobbs. However, for general purpose spectrophotometry, measurements are made over a large range of wavelengths, as with a scanning instrument or multiple filter unit. In this case, the varying splitting ratio of the beam splitter, which is strongly wavelength dependent, will ensure that LOGO will be large over some portion of the wavelength range, which can give rise to the measurement limitations relating to tracking errors and digitization requirements discussed above. Haller and Hobbs also recognized that the experimental apparatus required to perform high sensitivity spectrophotometry is subject to serious noise and drift problems.
Another noise canceling circuit is described by He in U.S. Pat. No. 5,742,200. This circuit functions with feedback similar to the Hobbs circuit, but it can also provide bias so that the background output can be adjusted to zero voltage. However, the bias voltage would have to be continuously adjusted to maintain balance over a broad wavelength range.
Such noise cancellation techniques will have applicability in special applications to cancel coherent noise. While it is possible to use either of the circuits described above (or others) to cancel coherent source noise in special circumstances, general spectrophotometry as used for routine chemical analysis and similar applications presents additional problems. Generally, the wavelength of the source must be variable, so that a laser cannot be used in general. Also, once the source noise is cancelled, thermal noise/drift becomes dominant, as will be described in detail below. Thus, there remains a need in the art. The present invention seeks to improve upon the state of the art of spectrophotometry.
The invention concerns improvements in spectrophotometry. Aspects of the invention may be used independently or together to increase the sensitivity of spectrophotometry. Exemplary preferred aspects and embodiments of the invention will be briefly summarized now.
One aspect of the invention is a spectrophotometer detection circuit. In this aspect of the invention, currents attributable to reference and sample beams are cancelled in the current mode. The detection circuit produces a first voltage proportional to the difference in currents and a second voltage proportional to one of the reference or sample beams. Both voltages are available to allow simultaneous measurement and analysis. Another aspect of the invention is a unique beam splitter configuration, which uses three beam splitters to ensure that the relative powers, phases, and polarizations of two beams derived from a single light source remains constant over a range of wavelengths. Another aspect of the invention concerns thermal stability. According to the invention, thermal conductivity is established among the housing and optical system components to promote equilibrium. One preferred embodiment has a unitary solid metal housing with a hollowed portion defined to mount and place optical system components. An additional aspect of the invention concerns optical filtering of the spectrophotometer source beam. In a preferred embodiment spectrophotometer, the optical source is isolated by making it external to other device components, and feeding the beam in through an optical fiber. Partially polarized light emerges from the optical fiber. The inventors have recognized dependence of the polarization effect upon ambient temperature, and the resultant potential differential drift in the optical system. A preferred embodiment spectrophotometer uses a holographic diffuser to reduce dependence of the beam splitting ratio upon varying polarization.
Recognition and identification of important noise sources in spectrophotometers forms an aspect of the invention contributing to the features and combinations of features in preferred embodiments. Many noise sources would not normally be considered in conventional spectrophotometry because the magnitude of particular noise sources dominates device performance. Thus, another aspect of the invention addresses the potential interferences caused by airborne particulates in the beam paths, bubbles and suspended particulates in liquids under study, and changing temperature at the glass/liquid interfaces in liquid cells. Another aspect of the invention addresses the potential interference caused by light reflected from the surfaces of the detectors.